Messenger RNA (mRNA) breakdown in bacteria can be defined by 3 parameters: by chemical or mass loss of the message, by the loss of capacity to serve as a template for protein synthesis, and by loss of potential (the ability of the message to load ribosomes which then translate it). The mRNA from the lactose operon (lac) is attacked by endonucleolytic cleavage into discrete sizes that can be identified by hybridization to defined segments of lac DNA; the same is true of the mRNA from the galactose operon (gal). Since these cleavage targets are intercistronic, we wish to determine if this cleavage is responsible for the inactivation of the distal message. Fragments of mRNA released by this cleavage will be collected from specific fractions after sucrose gradient centrifugation. Each will be tested for activity to direct synthesis of its specific enzyme in a cell-free system for enzyme synthesis. We hope to determine the nucleotide sequences at the 5' and 3' ends of the released messages. At the same time we shall determine the nucleotide sequences of the three ribosome loading sites of the lac mRNA. If the sequence of nucleotides at the ends of the released fragments correspond to the sequences within the ribosome loading sites, this will serve to identify the targets of the endonucleolytic cleavage. At the same time we are continuing studies to determine the role of translation and ribosomes in mRNA decay. Specific inhibitors of translation are being used in these studies. The decay of mRNA in strains carrying mutations for a specific ribonuclease are being examined in an effort to identify the enzymes that may be involved in the process. BIBLIOGRAPHIC REFERENCES: Lim, L.W. and Kennell, D. Role of endonucleolytic cleavage in decay of E. coli messenger RNA. Submitted to Molec. Gen. Genetics, November, 1976. Kennell, D. and Riezman, H. (1977). Transcription and translation initiation frequencies of the E. coli lac operon. J. Molec. Biol. in press.